Eadie-Hofstee plots of bosentan metabolism using human microsomes: the production of hydroxyl bosentan (A) and desmethyl bosentan (B). v and v/s represent the initial velocity of bosentan metabolism and metabolic clearance of bosentan, respectively. The initial velocity of these metabolites was calculated using the production volume obtained after 3-minute incubation. The solid line represents the fitted curve by nonlinear least-squares methods. Each point represents the mean ± S.D. The inset panel shows the “v vs. c” curve of bosentan metabolism using human microsomes.

Monte Carlo simulation of bosentan blood concentration profiles. The results of the Monte Carlo simulations that considered interindividual variability in Vmax,uptake, Km,uptake, PSdif, and CLmet and intraindividual variability in model 3. Observed mean and S.E. values of each dose are shown as closed circles and lines, and mean values of dose-normalized AUCs of each virtual study estimated from Monte Carlo simulation using model 3 are indicated as closed rectangles.

Tables

The kinetic parameters experimentally obtained from in vitro studies are presented as the mean ± S.D., unless otherwise indicated.

Parameters

Value

Source

Physiologic parameters

Body weight (kg)

78

Weber et al. (1996)

Hepatocellular space (g/kg)

6.69

Davies and Morris (1993)

Extrachepatic space (g/kg)

17.4

Adipose (g/kg)

142

Muscle (g/kg)

429

Skin (g/kg)

111

Blood flow rate

Liver (ml/min per kilogram)

20.7

Davies and Morris (1993)

Adipose (ml/min per kilogram)

3.72

Muscle (ml/min per kilogram)

10.7

Skin (ml/min per kilogram)

4.28

Tissue/blood concentration ratio

Adipose

0.121

Calculated from reported equations (Rodgers and Rowland, 2006)

Muscle

0.119

Skin

0.483

Kinetic parameters

Plasma unbound fraction

0.02

Weber et al. (1996), Dingemanse and van Giersbergen (2004)

Blood/plasma concentration ratio

0.6

CLr (l/h)

0.144

Vmax,uptake (pmol/min per 106 cells)

47.4 ± 18.6

Current study

Km,uptake (μM)

1.33 ± 1.34

PSdif,inf (pmol/min per 106 cells)

2.89 ± 0.46

Vmax,met,OH (pmol/min per milligram microsomal protein)

16.4 ± 1.75

Km,met,OH (μM)

6.40 ± 1.20

CLmet,OH,nonsaturable (μl/min per milligram microsomal protein)

0.158 ± 0.015

Vmax,met,DES (pmol/min per milligram microsomal protein)

7.53 ± 2.39

Km,met,DES (μM)

4.80 ± 2.61

CLmet,DES,nonsaturable (μl/min per milligram microsomal protein)

0.273 ± 0.025

fH

0.0696 ± 0.0068

CL,met,DES,nonsaturable, nonsaturable CLmet for the production of desmethyl bosentan; CL,met_OH,nonsaturable, nonsaturable CLmet for the production of hydroxyl bosentan; Vmax,met,DES, Vmax,met for the production of desmethyl bosentan; Vmax,met,OH, Vmax,met for the production of hydroxyl bosentan.

CL,met_OH,nonsaturable, nonsaturable CLmet for the production of hydroxyl bosentan; SF,met, scaling factors for Vmax for the production of desmethyl bosentan, Vmax for the production of hydroxyl bosentan, and CLmet; SF,transport, scaling factors for Vmax,uptake and PSdif; Vc, distribution volume of the central compartment; Vmax,met,DES, Vmax,met for the production of desmethyl bosentan; Vmax,met,OH, Vmax,met for the production of hydroxyl bosentan.

↵a Range is the constraint on the estimates for each parameter in model fitting to data.